1. Field of the Invention
The invention relates generally to the field of molecular biology and to methods of isolating and purifying recombinant proteins. The invention also includes DNA constructs engineered to include DNA encoding a desired polypeptide and a unique plant virus proteinase recognition site. Expression vectors for use in overproducing recombinant plant virus proteinase, particularly tobacco etch virus proteinase, are also part of the invention. The recombinant plant virus proteinase is unaffected by cell protease inhibitors, thereby permitting isolation of significantly improved yields of proteolytically sensitive polypeptides in the presence of added cell protease inhibitors. Methods of overproducing plant viral proteinases are also disclosed.
2. Description of Related Art
Isolation and purification of recombinant polypeptides expressed in bacterial and other hosts is of interest because these systems are capable of providing relatively large quantities of polypeptides having therapeutic or commercial value. Such systems are especially desirable where synthesis is expensive and time-consuming and/or the protein occurs naturally only in small quantities. Unfortunately, yields of recombinant polypeptides are frequently drastically reduced because of any one of several problems that may be encountered, including difficulties associated with isolation, purification and segregation.
Fusion proteins are frequently the method of choice for purifying proteins because of the benefit of allowing affinity purification and the increase in solubility that is frequently observed. There are, however, several disadvantages associated with release of the protein of interest from the carrier protein. The proteases employed for cleavage, typically thrombin and factor x, have recognition sites with low specificity so that the protein is often cleaved at several sites. Cleavage may result in several foreign amino acid residues attached to the protein, thereby altering the properties of the protein.
Several proteases not only lack selectively but are also relatively inefficient, so that yields of the cleaved protein are significantly decreased. In some cases, this is due to impurities which may either partially inactivate the protease or, more commonly, themselves have nonselective proteolytic activity.
A selective tobacco etch virus proteinase cleavage site said to be useful for expressing fusion proteins has been disclosed (Jarvis and Carrington, 1993). Unfortunately, specific constructs disclosed were stated to be useful only for expression in insect cells.
Fusion proteins tend to be more soluble than the single protein, contributing to higher yields and simpler purification; however, there are often problems in removing the fusion "partner". This is generally accomplished by cleaving the two proteins, provided there is a selective cleavage site that will leave the desired protein intact. Often, however, it is difficult to obtain the desired polypeptide free of contaminating segments of the fusion partner.
Numerous recombinant systems are available for promoting synthesis of fusion proteins in a bacterial host cell. Such fusion proteins may be generated as single chain polypeptides following translation of appropriately joined DNA segments. Fusion proteins incorporate the protein of interest, a carrier protein and, typically, a polylinker sequence between the two proteins. Carrier proteins may be selected on the basis of transport characteristics to assure that the fusion protein is secreted into either the periplasmic space or the growth medium on the basis of solubility. Regardless of the amount of fusion protein expressed, isolation and purification of the desired polypeptide is frequently difficult and inefficient.
One approach to the isolation problem has been to engineer fusions of a desired polypeptide with a carrier protein having specific binding properties; for example, binding to affinity columns that incorporate antibodies, chelates, metals etc. Isolation is thereby facilitated and product yields improved. Recombinant eukaryotic proteins have been synthesized from gene fusions of foreign proteins with the carbohydrate-recognition domain (CRD) of the galactose-specific rat hepatic lectin (Taylor and Drickamer, 1991). These fusion proteins are isolated affinity chromatography, then cleaved at a proteinase-sensitive linker.
A popular carrier protein is Staphylococcus aureus protein A. A recombinant protein is first isolated by affinity chromatography, which binds protein A, then separated by domain-specific enzymatic cleavage with chymotrypsin while immobilized on the affinity column. The affinity column is specific for protein A which binds to the F.sub.c portion of certain subclasses of human immunoglobulins, including IgG. A detailed description of the use of protein A in this manner is provided in International Patent Application Number WO 84/03103 incorporated herein by reference.
Several proteins have been isolated and purified using this general scheme, including DNA-binding domain of the glucocorticoid receptor (Dahlman, et al., 1989). Eukaryotic proteins expressed in Escherichia coli have been isolated from crude bacterial extracts by affinity chromatography on glutathione agarose when the protein is expressed as a polypeptide in frame with glutathione-S-transferase (Smith and Johnson, 1988). The vector system usually incorporates specific protease cleavage sites to facilitate proteolysis for cleavage of the desired protein from glutathione-S-transferase. Modification of the vector in one instance to include a glycine-rich linker has resulted in increased thrombin cleavage efficiency for several fusion proteins (Guan and Dixon, 1991).
Despite improved yields and facilitated purification when employing these carrier "affinity" protein fusions, expressed fusion proteins are frequently seriously damaged or even destroyed by endogenous bacterial proteases. Yields are therefore low despite an efficient expression vector. This is particularly a problem for protease sensitive small proteins or peptides. Addition of cell protease inhibitors to crude bacterial extracts may alleviate proteolysis; however, cleavage of a foreign protein from its carrier protein becomes a problem because the inhibitors will typically inactivate added cleavage proteases that are commonly employed for this purpose. The presence of added proteases and protease inhibitors also complicates purification because of additional species and product dilution.
As previously mentioned, a significant problem may be cleavage of the expressed foreign protein or peptide by the proteinase employed (e.g., thrombin, factor x), employed to release the desired polypeptide from the carrier protein. These spurious cleavages are the result of the limited specificity displayed by the proteinases frequently employed.
Finally, in the isolation of recombinant proteins, spurious amino acids are frequently attached to the cleaved polypeptide product. These amino acids are typically present when a linker is cleaved, and the unrelated amino acids may have an effect on the properties of the isolated protein. This may be critical for proteins produced for human therapeutics. Ideally, one seeks efficient cleavage to produce pure native protein free of extraneous amino acid short sequences or residues.